Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of mycobactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)

This European Standard specifies a test method and the minimum requirements for mycobactericidal activity
of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when
diluted with hard water or in the case of ready-to-use-products with water. Products can only be tested
at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and
interfering substance.
This European Standard applies to products that are used in the veterinary area i.e. in the breeding,
husbandry, production, transport and disposal of all animals except when in the food chain following death
and entry to the processing industry.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances
under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test (Annex F).

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der mykobakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Diese Europäische Norm legt ein Prüfverfahren (Phase 2, Stufe 1) und die Mindestanforderungen an die mykobakterizide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die in hartem Wasser eine homogene, physikalisch stabile Zubereitung bilden. Diese Europäische Norm gilt für Produkte für den Veterinärbereich, d. h. bei deren Verwendung bei der Aufzucht, Haltung, Produktion und beim Transport von Tieren sowie bei der Tierkörperbeseitigung, außer wenn die Tiere nach der Tötung durch Zuführung in die weiterverarbeitende Industrie in die  Nahrungsmittelkette eintreten. Bei Anwendung dieser Europäischen Norm ist die Bestimmung der mykobakteriziden Wirkung eines unverdünnten Produktes nicht möglich, da immer eine bestimmte Verdünnung durch Zugabe der Impfkultur und der Belastungssubstanz erfolgt. Produkte können nur bis zu einer Konzentration von 80 % geprüft werden.

Désinfectants et antiseptiques chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité mycobactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (Phase 2, étape 1)

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za ocenjevanje mikobaktericidnega delovanja kemičnih razkužil in antiseptikov, ki se uporabljajo v veterini - Preskusna metoda in zahteve (faza 2, stopnja 1)

General Information

Status
Withdrawn
Publication Date
30-Nov-2004
Withdrawal Date
19-Nov-2012
Current Stage
9900 - Withdrawal (Adopted Project)
Start Date
09-Nov-2012
Due Date
02-Dec-2012
Completion Date
20-Nov-2012

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der mykobakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Désinfectants et antiseptiques chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité mycobactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (Phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of mycobactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)11.220VeterinarstvoVeterinary medicine11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 14204:2004SIST EN 14204:2004en,fr,de01-december-2004SIST EN 14204:2004SLOVENSKI

STANDARD
SIST EN 14204:2004

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14204June 2004ICS 11.080.20English versionChemical disinfectants and antiseptics - Quantitative suspensiontest for the evaluation of mycobactericidal activity of chemicaldisinfectants and antiseptics used in the veterinary area - Testmethod and requirements (phase 2, step 1)Antiseptiques et désinfectants chimiques - Essai quantitatifde suspension pour l'évaluation de l'activitémycobactéricide des antiseptiques et des désinfectantschimiques utilisés dans le domaine vétérinaire - Méthoded'essai et prescriptions (Phase 2, étape 1)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Suspensionsversuch zur Bestimmung dermykobakteriziden Wirkung chemischer Desinfektionsmittelund Antiseptika für den Veterinärbereich - Prüfverfahrenund Anforderungen (Phase 2, Stufe 1)This European Standard was approved by CEN on 20 February 2004.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36

B-1050 Brussels© 2004 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14204:2004: ESIST EN 14204:2004

EN 14204:2004 (E) 2 Contents Page Foreword..............................................................................................................................................................3 Introduction.........................................................................................................................................................4 1 Scope......................................................................................................................................................5 2 Normative references............................................................................................................................5 3 Terms and definitions...........................................................................................................................5 4 Requirements.........................................................................................................................................6 5 Test method............................................................................................................................................6 Annex A (normative)

Test for validation of dilution-neutralization method..............................................18 Annex B (informative)

Homogenization of mycobacterial test suspensions............................................20 Annex C (informative)

Neutralizers................................................................................................................21 Annex D (informative)

Example of a typical test report...............................................................................22 Annex E (informative)

Corresponding referenced strains..........................................................................24 Annex F (informative)

Information on the application and interpretation of European Standards on chemical disinfectants and antiseptics.......................................................................................25 Bibliography......................................................................................................................................................27

SIST EN 14204:2004

EN 14204:2004 (E) 3 Foreword This document (EN 14204:2004) has been prepared by Technical Committee CEN/TC 216 “Chemical disinfectants and antiseptics”, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by December 2004, and conflicting national standards shall be withdrawn at the latest by December 2004. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and the United Kingdom. SIST EN 14204:2004

EN 14204:2004 (E) 4 Introduction This European Standard describes a suspension test for establishing whether a chemical disinfectant or antiseptic in the veterinary field has or does not have mycobactericidal activity under the laboratory conditions, defined by this European Standard, which influence the action of disinfectants in practical use. The type and level of interfering substance can be selected as well as contact times and temperatures in addition to the levels specified in order to support recommendations for use under particular conditions. The method involves neutralization of the mycobactericidal activity at the moment of sampling by dilution into a previously validated neutralizer. The conditions that shall be tested are intended to cover general purposes and to allow reference between laboratories and product types. For some applications, however, the recommendations of use of a product may differ and therefore additional test conditions need to be used. SIST EN 14204:2004

EN 14204:2004 (E) 5 1 Scope This European Standard specifies a test method and the minimum requirements for mycobactericidal activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water or — in the case of ready-to-use-products — with water. Products can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance. This European Standard applies to products that are used in the veterinary area – i.e. in the breeding, husbandry, production, transport and disposal of all animals except when in the food chain following death and entry to the processing industry. NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1 test (Annex F). 2 Normative references

The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

EN 1656, Chemical disinfectants and antiseptics – Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field - Test method and requirements (phase 2/step 1). EN 12353, Chemical disinfectants and antiseptics – Preservation of microbial strains used for the determination of bactericidal and fungicidal activity. 3 Terms and definitions For the purposes of this European Standard, the following terms and definitions apply. 3.1 product chemical agent or formulation used as a chemical disinfectant or antiseptic [EN 1040] 3.2 mycobactericide product that kills mycobacteria under defined conditions NOTE The adjective derived from "mycobactericide" is "mycobactericidal". 3.3 mycobactericidal activity capability of a product to produce a reduction in the number of viable mycobacterial cells of relevant organisms under defined conditions SIST EN 14204:2004

EN 14204:2004 (E) 6 4 Requirements The product diluted in hard water when tested, in accordance with clause 5, shall demonstrate at least a lg reduction in viable counts of 4, when the test organism is Mycobacterium avium. The test is carried out under simulated low level soiling conditions (3.0 g/l bovine albumin) or simulated high level soiling conditions (10 g/l yeast extract and 10 g/l bovine albumin) according to its practical applications and under the required test conditions 10 °C, 60 min, 1 referenced strain. Where appropriate additional and optional contact times of 1 min ± 5 s, 5 min ± 10 s, 15 min ± 10 s, 30 min ± 10 s and 120 min ± 10 s and additional and optional temperatures of 4 °C, 20 °C and 40 °C are specified. 5 Test method 5.1 Principle 5.1.1 A prepared sample of the product under test diluted in hard water is added to a test suspension of mycobacterial cells in a solution of interfering substances. The mixture is maintained at 10 °C ± 1 °C. At a contact time of 60 min ± 10 s an aliquot is taken ; the mycobactericidal action in this portion is immediately neutralized or suppressed by a validated method. The number of surviving mycobacteria in each sample is determined and the reduction in viable counts calculated. Preliminary tests leading to the choice of the inactivation method should be carried out before the actual test. However, it is necessary to check the neutralization of the carry over in parallel with the actual test. 5.2 Materials and reagents 5.2.1 Test organism 5.2.1.1 The mycobactericidal activity shall be evaluated using the following strain :  Mycobacterium avium ATCC 157691) NOTE See Annex E for corresponding strain numbers in some other culture collections. 5.2.1.2 If required for specific applications additional strains may be chosen and must be noted in the test report. Their suitability for supplying inocula of sufficient concentration shall be verified. If the additional strains tested are not classified at a reference centre, identification characteristics shall be given. In addition they shall be held by the testing laboratory under a reference for 5 years. If additional strains do not grow on the medium (see 5.2.2.3) and/or cannot be used with diluent (see 5.2.2.4) additional media shall be used and shall be reported as well as additional cultivation conditions.

1) ATCC 15769 is the collection number of strain supplied by the American Type Culture Collections. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named.

Corresponding strains supplied by other culture collections may be used if they can be shown to lead to the same results. SIST EN 14204:2004

EN 14204:2004 (E) 7 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. NOTE To improve the reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturer’s instructions relating to the preparation of these products should be rigorously followed. 5.2.2.2 Water The water shall be free from substances that are toxic or inhibiting to the mycobacteria. It shall be freshly glass distilled and not demineralised water. Sterilize in the autoclave (see 5.3.2.1a). NOTE 1 If the water is sterilized during the sterilization of the reagents this is not necessary. NOTE 2 If distilled water of adequate quality is not available, water for injectable preparations (European Pharmacopaeia) can be used. 5.2.2.3 Middlebrook and Cohn 7H10 medium + 10 % OADC (reported as 7H10 in the text). For performance of viable counts :  middlebrook 7H10 agar 19 g ;  glycerol 5 ml ;  water (see 5.2.2.2) to 895 ml ; Heat to boiling to dissolve completely. Sterilize for 10 min in the autoclave and cool to 50 °C to 55 °C. Add 100 ml Middlebrook OADC enrichment under aseptic conditions. Final pH = 6,6 ± 0,2 at 25 °C. 5.2.2.4 Diluent Tryptone Sodium Chloride Solution :  tryptone, pancreatic digest of casein 1,0 g ;  sodium Chloride (NaCl) 8,5 g ;  water (see 5.2.2.2) to 1 000 ml ; Sterilize in the autoclave (see 5.3.2.1). After sterilization the pH of the medium shall be equivalent to 7,0 ± 0,2, when measured at 20 °C. SIST EN 14204:2004

EN 14204:2004 (E) 8 5.2.2.5 Neutralizer The neutralizer shall be validated for the product under test in accordance with Annex A. The neutralizer shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in Annex C. 5.2.2.6 Phosphate buffer 0,25 mol/l  Potassium Dihydrogen Phosphate (KH2PO4) 34 g ;  water (see 5.2.2.2) to 500 ml. Adjust the pH to 7,2 ± 0,1 with 1 mol/l sodium hydroxide (NaOH) ;  water (see 5.2.2.2) to 1 000 ml. Sterilize in the autoclave. 5.2.2.7 Hard water for dilution of products For the preparation of 1 l of hard water, the procedure is as follows:  prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave [5.3.2.1a)]. Store the solution at 2 °C to 8 °C for no longer than one month; If necessary make up to 1 000 ml with water (5.2.2.2) under aseptic conditions.  prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution at 2 °C to 8 °C for no longer than one week;  place 600 ml to 700 ml of water (5.2.2.2) in a 1000 ml volumetric flask (5.3.2.12) and add 6,0 ml of solution A, then 8,0 ml of solution B. Mix and dilute to 1000 ml with water (5.2.2.2). The pH of the hard water shall be 7,0 ± 0,2, when measured at 20 ± 1°C (5.3.2.4). If necessary, adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl). The hard water shall be freshly prepared under aseptic conditions and used within 12 hours. NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces a different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube. 5.2.2.8 Interfering substances 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product. The interfering substance shall be sterile and prepared at 10 times its final concentration in the test. The composition shall be noted in the test report (see 5.8). SIST EN 14204:2004

EN 14204:2004 (E) 9 5.2.2.8.2 Bovine albumin solution for low level soiling conditions  Dissolve 3 g of bovine albumin (Cohn fraction V for Dubos Medium) in 90 ml of water (see 5.2.2.2) in a 100 ml volumetric flask. Make up to the mark with water (see 5.2.2.2) ;  sterilize by membrane filtration. The final concentration of the bovine albumin in the test procedure (see 5.5.2) is 3 g/l. 5.2.2.8.3 Albumin/yeast extract mixture for high level soiling conditions a) Dissolve 50 g yeast extract powder in 150 ml of water (see 5.2.2.2) in a 250 ml volumetric flask and allow foam to collapse. Make up to the mark with water (see 5.2.2.2). Transfer to a clean dry bottle and sterilize in the autoclave (see 5.3.2.1). Allow to cool to 20 °C ± 5 °C ; b) pipette 25 ml of this solution into a 50 ml volumetric flask and add 10 ml of water (see 5.2.2.2). Dissolve 5 g of the bovine albumin (Cohn fraction V for Dubos Medium) in the solution in the flask with shaking and allow foam to collapse. Make up to the mark with water (see 5.2.2.2) sterilize by filtration and keep in 10 ml portions in a refrigerator (see 5.3.2.15) until use. The final concentration in the test procedure (see 5.5.2) is 10 g/l yeast extract and 10 g/l bovine albumin. 5.2.2.9 7H9 broth + 10 % ADC (reported as 7H9 broth in the text)  Ammonium Sulphate 0,5 g  L. glutamic Acid 0,5 g  Sodium Citrate 0,1 g  Pyridoxine 0,001 g  Biotin 0,0005 g  Disodium phosphate 2,5 g  Monopotassium Phosphate 1 g  Ferric ammonium Citrate 0,04 g  Magnesium Sulphate 0,05 g  Calcium Chloride 0,0005 g  Zinc Sulphate 0,001 g  Copper Sulphate 0,001 g Dissolve 4,7 g in 900 ml water (see 5.2.2.2) (containing 2 ml glycerol or 0,5 g Tween 80 2) if desired) sterilize at 121 °C to 124 °C for 10 min. Aseptically add 100 ml Middlebrook OADC enrichment to the medium at 45 °C. Final pH = 6,6 ± 0,2 at 25 °C.

2) Analytical quality, non-hydrolized in accordance with European Pharmacopaeia volume 1. TWEEN 80  is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 14204:2004

EN 14204:2004 (E) 10 5.3 Apparatus and glassware 5.3.1 General Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods: a) by moist heat, in the autoclave [5.3.2.1a)]; b) by dry heat, in the hot air oven [5.3.2.1b)]. 5.3.2 Usual microbiological laboratory equipment2) and, in particular, the following: 5.3.2.1 Apparatus for sterilization a) For moist heat sterilization, an autoclave capable of being maintained at 121+ C

30°+for a minimum holding time of 15 min ; b) for dry heat sterilization, a hot air oven capable of being maintained at 180 0+ 5°C for a minimum holding time of 30 min, at 170 0+ 5°C for a minimum holding time of 1 h or at 160 0+ 5°C for a minimum holding time of 2 h ; c) membrane filtration apparatus suitable for use with filters of diameter 47 mm to 50 mm of 0.22 µm pore size for media sterilization. 5.3.2.2 Water baths, capable of being controlled at 4 °C ± 1 °C, 10 °C ± 1 °C, 20 °C ± 1 °C and 40 °C ± 1 °C and 45 °C ± 1 °C. 5.3.2.3 CO2 Incubator, capable of being controlled at either 36 °C ± 1 °C or 37 °C ± 1 °C. An incubator at 36 °C ± 1 °C or 37 °C ± 1 °C may be used if a CO2 incubator is not available. 5.3.2.4 pH-meter, having an accuracy of calibration of 0,1 pH units at 25 °C. 5.3.2.5 Stopwatch. 5.3.2.6 Electromechanical agitator, e.g. Vortex  mixer3). 5.3.2.7 Containers, test tubes, flasks or bottles of suitable capacity. 5.3.2.8 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0.1 ml. Calibrated automatic pipettes may be used. 5.3.2.9 Petri dishes of size 90 mm to 100 mm. 5.3.2.10 Glass beads (Diameter : 3 mm to 4 mm). 5.3.2.11 Volumetric flasks (calibrated at 20 °C). 5.3.2.12 Mechanical shaker. 5.3.2.13 Plastic screw cap tubes (50 ml).

2) Disposable Sterile Equipment is an acceptable alternative to reusable glassware. 3) Vortex  is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 14204:2004

EN 14204:2004 (E) 11 5.3.2.14 Plastic disposable 10 µl loops. 5.3.2.15 Refrigerator, capable of being controlled at between 2 °C and 8 °C. 5.3.2.16 High Speed homogenizer, e.g. Potter  apparatus4) 5.4 Preparation of mycobacterial suspension and test solutions 5.4.1 Mycobacterial suspension 5.4.1.1 Stock culture of test organism Use the following method. NOTE It is envisaged that a specific part of the European standard EN 12353 "Chemical disinfectants and antiseptics - Preservation of microbial strains used for determination of activity" will replace this method in due course. A stock culture can be prepared by subcultivating the lyophilized strain (see 5.2.1) on plates with 7H10 medium (see 5.2.2.3). Incubate at 36 °C ± 1 °C or 37 °C ± 1 °C for at least 21 days in a CO2 incubator. If a CO2 incubator is not used plates shall be protected from drying by sealing with insulating tape or packing them into polyethylene bags. Cultures are recovered from 7H10 plates using 7H9 broth (see 5.2.2.9), divided into 1 ml portions and stored at –70 °C for prolonged storage. For short storage the stock culture is maintained on separate 7H10 slopes in a refrigerator (see 5.3.2.15) for a maximum of 4 weeks. 5.4.1.2 Working culture of the organism In order to prepare a working culture of Mycobacterium avium (see 5.2.1.1) subculture from the stock culture (see 5.4.1.1) on 7H10 medium (see 5.2.2.3) and incubate at 36 °C ± 1 °C (or 37 °C ± 1 °C) (see 5.3.2.3). If a CO2 incubator is not used, plates shall be placed into polyethylene bags or sealed with insulating tape to prevent drying of the medium. After 21 days prepare a second subculture from the first subculture and incubate for 21 days. NOTE The first and/or the second subculture is the working culture(s). 5.4.1.3 Mycobacterial test suspension Prepare a suitable homogeneous suspension from the working culture (see 5.4.1.2) following one of the methods given in Annex B (see B.1 or B.2) other methods of homogenization can be used provided that the cfu number obtained is appropriate and stable during the time of the test and that microscopic examination shows that the suspension is homogenously dispersed, without aggregates. For counting the mycobacterial test suspension prepare 10-6 10-7 and 10-8 dilutions of the test suspension using diluent (see 5.2.2.4). Mix (see 5.3.2.6). Take a sample of 0,5 ml of each dilution in duplicate and spread each sample on separate Petri dishes containing 18 ml to 20 ml of 7H10 medium (see 5.2.2.3). 5.4.1.4 Counting of mycobacterial test suspension Incubate the plates at 36 °C + 1 °C (or 37 °C + 1 °C) (see 5.3.2.3) for 21 days (see 5.4.1.1). Discard any plates which are not countable. Count the plates and determine the number of colony forming units.

4) Potter  Apparatus is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 14204:2004

EN 14204:2004 (E) 12 Incubate the plates for a further 7 days. Do not recount plates which no longer show well separated colonies. Recount the remaining plates. Determine the higher number of colonies Vc for each plate. Calculate the number of cfu/ml5) in the test suspension (N) using the method given in 5.6.1.1. 5.4.2 Product test solution Details of samples of the product as received shall be recorded. Product test solutions shall be prepared in hard water (see 5.2.2.7) at three different concentrations to include one concentration in the active range and one concentration in the non-active range. The concentration of the product test solution shall be 1.25 times the required test concentration. NOTE The product as received may be used as one of the product test solutions. For solid products, dissolve the product as received by weighing at least 1 g ± 10 mg of the product in a volumetric flask and filling up with hard water (see 5.2.2.7). Subsequent dilutions shall be prepared in volumetric flasks (see 5.3.2.11) on a volume/volume basis in hard water (see 5.2.2.7). For liquid products, dilutions of the product shall be prepared in hard water (see 5.2.2.7) on a volume/volume basis using volumetric flasks (see 5.3.2.11). For products supplied in a ready to use state, water (see 5.2.2.2) shall be used to prepare dilutions. The product test solutions shall be prepared freshly and used within 60 min. If a shorter time is specified in the manufacturer’s recommended conditions of use, this time should be followed. The concentration of the product stated in the test report shall be the test concentration. Record the test concentration in terms of mass per volume or volume per volume. 5.5 Procedure 5.5.1 Choice of experimental conditions The selection of contact temperature, contact time and interfering substances shall be carried out according to the practical use considered for the product (see 4) as follows : a) temperature : θ in °C ;  the temperature to be tested is 10 °C ± 1 °C ;  the additional temperatures may be chosen from 4 °C ± 1 °C or 20 °C ± 1 °C or 40 °C ± 1 °C ; b) contact time : t in min ;  the contact time to be tested is 60 min ± 10 s ;  the additional contact times may be chosen from 1min ± 5 s; 5 min ± 10 s ; 10 min ± 10 s, 15 min ± 10 s, 30 min ± 10 s or 120 min ± 10 s ; c) strain ;  strain shall be as stated in 5.2.1.1 ;

5) cfu/ml = colony forming units per ml. SIST EN 14204:2004

EN 14204:2004 (E) 13 d) interfering substance ;  the obligatory interfering substance to be tested is 3.0 g/l bovine albumin (see 5.2.2.8.2) under simulated clean conditions or 10.0 g/l albumin/yeast extract mixture (see 5.2.2.8.3) under simulated dirty conditions according to practical applications (see 4) ; e) the obligatory test organism is Mycobacterium avium (see 5.2.1). If a precipitate is formed in the experimental conditions it should be recorded in the test report. Each selected experimental condition (θ °C, t, strains and interfering substance) shall be validated in accordance with Annex A. 5.5.2 Test procedure 5.5.2.1 General To determine a suitable neutralizer the following procedure shall be adopted. Carry out the validation of the dilution-neutralization method (see A.4) using a suitable neutralizer, chosen according to laboratory experience and published data. If this neutralizer is not valid, repeat the validation test using an alternative neutralizer containing a combination of polysorbate 80 30 g/l, L-histidine 1 g/l, lecithin 3 g/l, sodium thiosulphate 5 g/l, saponin 30 g/l in either diluent (see 5.2.2.4) or in phosphate buffer 0.25 mol/l at 1 % (see Annex C). The inactivation of the mycobactericidal activity of the product shall be validated for the test strain and for each of the chosen experimental conditions (see 5.5.1). 5.5.2.2 Dilution-neutralization method 5.5.2.2.1 General Prior to testing, equilibrate all reagents (product test solutions, mycobacterial test suspension interfering substances) to the test temperature ± 1 °C using the water bath (see 5.3.2.2). Check that the temperature of the reagents is stabilized at the test temperature ± 1 °C. The neutralizer and water (see 5.2.2.2) shall be equilibrated at a temperature of 20 °C ± 1 °C. 5.5.2.2.2 Test procedure for mycobactericidal activity of products Pipette 1,0 ml of the interfering substance (see 5.2.2.8) into a test tube. Mix (see 5.3.2.6) and add 1,0 ml of the mycobacterial test suspension containing 3 × 108 to 8 × 108 cfu/ml (see 5.4.1.3). Start the stopwatch immediately, mix (see 5.3.2.6), and place the test tube in the water bath (see 5.3.2.4) at 10 °C ± 1 °C for 2 min ± 10 s. At the end of this time, add 8,0 ml of one of the product test solutions. Restart the stopwatch, mix (see 5.3.2.6) and place the test tube in a water bath (see 5.3.2.4) controlled at 10 °C ± 1 °C for 60 min ± 10 s. NOTE When adding mycobacterial suspensions care should be taken to avoid touching the upper part of the test tube sides. Just before the end of the contact time, mix (see 5.3.2.6). At the end of the contact time pipette 1,0 ml of the test mixture into a tube containing 8,0 ml neutralizer (see 5.2.2.5) and 1,0 ml water (see 5.2.2.2). Mix (see 5.3.2.8) and place in a water bath (see 5.3.2.2) controlled at 20 °C ± 1 °C. SIST EN 14204:2004

EN 14204:2004 (E) 14 After a neutralization time of 5 min ± 10 s, prepare a ten-fold dilution of the neutralized mixture in the diluent (5.2.2.4). Take a sample of 0.5ml of the neutralized mixture, and the ten-fold dilution in duplicate and spread each 0.5ml into separate petri dishes (5.3.2.9) containing 18ml to 20 ml 7H10 medium (5.2.2.3). Perform this procedure using the other product test solutions. 5.5.2.2.3 Counting the test mixture Incubate the plates at 36 °C + 1 °C (or 37 °C + 1 °C) (see 5.3.2.3) for 21 days. Discard any plates which are not countable. Count the plates and determine the number of colony forming units for each plate Incubate the plates for a further 7 days. Do not recount any plates which no longer show well-separated colonies. Recount the remaining plates. Det

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